Practice 4 Hemal biology. Department of parasitology, AHUM

Transcription

1 Practice 4 Hemal biology Department of parasitology, AHUM

2 Definition Hemal biology:study of normal and pathologic aspects of blood

3 Contents 1. blood components 2. hematopoiesis 3. blood function 4. white blood cell count 5. White blood cell differential 6. homework 7. case study

4 1. Blood components Adults have 4-6 L of blood A liquid connective tissue plasma matrix of blood a clear, light yellow fluid formed elements - blood cells and cell fragments red blood cells, white blood cells, and platelets

5 1. Blood components Blood cells (formed elements) suspended in plasma

6 Withdraw blood Centrifuge Plasma (55% of whole blood) Buffy coat: leukocytes and platelets (<1% of whole blood) Erythrocytes (45% of whole blood) Hematocrit Formed elements

7 Components of blood

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9 1. Blood components plasma (55%): water (90% of total plasma) protein (7%) albumen, globulin (antibodies), hormones, enzymes, cytokines blood coagulation factor: prothrombin, thrombin, fibrinogen other (3%) nutrients: fats, vitamins, carbohydrates (glucose) electrolytes gases: oxygen, carbon dioxide metabolic waste products: creatinine, urea nitrogen, uric acid, etc.

10 1. Blood components formed elements (45%) red blood cell (RBC, erythrocyte): male: /µl, female: /µl hemoglobin (Hb, male: 12-16g/dl, female:11-15g/dl, newborn:17-20g/dl) hematocrit (Hct, male: 42-52%, female: 37-47%): reticulocyte (Ret, immature erythrocyte): adult: %, newborn: % Mean Corpuscular Volume: MCV, fl Mean Cellular Haemoglobin Concentration: MCHC, g/dl

11 1. Blood components Mean Corpuscular Volume (MCV): MCV (fl) = Hct (%) 10 RBC (x 10 6 /µl) Mean Cellular Haemoglobin Concentration (MCHC): Hb (g/dl) MCHC (g/dl) = 100 Hct (%)

12 1. Blood components white blood cell (WBC, leukocyte): /µl granulocyte: granules, lobed nuclei, phagocytic neutrophil: 50-70% eosinophil: 0.5-3% basophile: 0-1% agranulocyte lymphocyte: 20-30% monocyte: 3-8% Platelet (thrombocytes): /µl

13 2. Hematopoiesis bone marrow: formation of blood cells occurs mostly in red bone marrow in adult. All cells arise from same blood stem cell (pluripotent hematopoietic stem cell), then become colonyforming units (CFUs), precursor cells, and mature cell In certain cases, immature and/or abnormal forms of the cells may be present in the blood and may be detected with a blood differential, so further bone marrow aspiration may be necessary to give an accurate diagnosis (metastatic or hematological malignancies)

14 2. Hematopoiesis

15 3. Blood function protection inflammation limit spread of infection destroy microorganisms and cancer cells neutralize toxins initiate clotting transport: nutrients, oxygen, carbon dioxide, hormones, enzymes, etc regulation: regulates fluid balance, acid-base balance, the temperature of the body

16 3. Blood function RBC carry oxygen via hemoglobin from lungs to peripheral tissues and carbon dioxide away from peripheral tissues to lungs reticulocyte: number increases in hemolytic anemia, decreases in aplastic anemia, radiation and chemotherapy

17 3. Blood function WBC leucocytes are an important part of the body's immune system and also have a role in inflammation, allergic responses, and protect against pathogens, abnormal cells (cancer cells), remove toxins and wastes

18 3. Blood function Neutrophils phagocytize and provide a first line of defense in acute infections increase in bacterial infection phagocytize bacteria and fungi release antimicrobial chemicals

19 3. Blood function lymphocytes lymphocytosis: viral infections, antigen stimulation lymphocytopenia: early stages of bacteria infection

20 3. Blood function Monocytes increase in viral infections and inflammation differentiate into macrophages and dendritic cells in the tissue phagocytize pathogens and debris present antigens to activate other immune cells take part in tissue repair

21 3. Blood function Eosinophils increase in parasitic infection (release enzymes destroy parasites) or allergic reaction phagocytize antigen-antibody complexes, allergens

22 3. Blood function Basophils increase in chicken pox, sinusitis, diabetes and immunolgically mediated hypersensitivity reactions secrete histamine, cytokines, heparin, inflammatory chemicals

23 3. Blood function Platelets hemostasis (vascular spasm, platelet plug, coagulation): blood clotting and blood vessel repair phagocytize bacteria attract WBCs to sites of inflammation

24 4. WBC count significance of WBC count diagnose an infection, immune disorders (autoimmune disorder, immune deficiency, and inflammation), blood disorders (anemia, leukemia), etc monitor the progression of conditions such as those named above monitor the body's response to various treatments

25 4. WBC count objective of the experiment learn to use hemocytometer and master the method of cell counting requirements for the experiment 0.02M acetic acid, alcohol cotton ball rubber leash, puncture needle, vacuum anticoagulant tube, cuvette a clean hemocytometer, cover slip, pipetting tube, microscope

26 4. WBC count protocol of the experiment draw blood from ulnar vein with a vacuum anticoagulant tube, mix sample well by inversion take 0.02M acetic acid 380 μl into a cuvette take blood 20 μl into the cuvette shake the cuvette to mix the blood and acetic acid get a clean hemocytometer, place the cover slip onto the counting gab, put the tip of the pipetting tube close to the edge of the cover slip, put a drop of solution into the crevice between the clivue and cover slip, to make it infiltrate to the counting gab put the microscope on low power objective, count the number of the cells in the 4 big squares of each corner of the counting gab count one by one, if the cells are on the lines count the upper but not the lower, the left but not the right

27 4. WBC count calculate the number of white blood cells per milliliter using the following equation: WBCs(number/ml)= (total number of cells in the four big squares/four) , 000 normal value adult: /µl child: /µl newborn: /µl

28 4. WBC count

29 4. WBC count High white blood cell count Definition: a high white blood cell count (leukocytosis): the count more than 10,000 white blood cells per microliter of blood Causes Infections: bacteria, viruses, fungi and parasites Inflammation or inflammatory conditions: rheumatoid arthritis, vasculitis or inflammatory bowel disease Allergic responses (e.g., allergies, asthma) Leukemia, myeloproliferative neoplasms: causing abnormally high production of WBCs Tissue necrosis (e.g., such as trauma, burns, surgery or heart attack) Pregnancy in the final month and labor Severe emotional or physical stress Intense exercise Drug (e.g., corticosteroids, epinephrine)

30 Low white blood cell count Definition: a low white blood cell count (leucopenia): the count lower than 4,000 white blood cells per microliter of blood. Causes Bone marrow damage and disorders Autoimmune disorders the body attacks and destroys its own WBCs (e.g., Systemic lupus erythematosus) Overwhelming infections that use up white blood cells faster than they can be produced (e.g., Sepsis) Diseases of the immune system, such as HIV, which destroy T lymphocytes, immune deficiency Malnutrition (Vitamin B12 or folate deficiency) Hypersplenism 4. WBC count Parasitic diseases (leishmaniasis)

31 5. White blood cell differential significance of the experiment WBC differential: estimates percentages of each type of WBCs a WBC differential determines the specific types of WBCs that are affected, and give further clues as to the cause of some diseases identify the presence of abnormal formed elements to help make a diagnosis

32 5. White blood cell differential objectives of the experiment realize the principle of slide staining with Wight's stain master the appropriate technique for staining a blood smear with Wight's stain learn how to observe the formed elements present in a slide, identify the presence of abnormal populations of blood cells and conduct a WBC differential count

33 5. White blood cell differential principle of the experiment Wright's stain is made by mixing methylene blue dye with eosin in a methanol diluent basic components of the cell, such as hemoglobin or certain inclusions or granules, will unite with the acidic portion of the stain, eosin, and are said to be eosinophilic. These components are stained varying shades of pink or red acidic cell components, such as nucleic acids, reactive cytoplasm, etc. take up the basic dye components, methylene blue, and stain blue or purple buffer ph is

34 5. White blood cell differential requirements for proper smear preparation Wright's stain, PBS buffer, rosin oil, zylene, alcohol cotton ball rubber leash, puncture needle, vacuum anticoagulant tube clean glass slide, spreader slide and coverslip, red pencil, microscope

35 5. White blood cell differential protocol of the experiment place a drop of blood at the right side of the slide place the spreader slide onto the lower slide in front of the blood drop, and pull the slide back until it touches the drop push the spreader slide forward at approximately a 30 degree angle, using a rapid, even motion when the smear is completely dried, mark the stained area using a red pencil completely cover the marked area with enough Wright's stain and keep it for 1-2 minutes add PBS buffer onto the blood smear slide, after 5 minutes, wash the slide for 30 seconds with tap water allow slide to dry at room temperature before examination observe under a microscope with 100X objective observe the formed elements, and calculate the percentage of each type of leukocytes found in the slide

36 5. White blood cell differential

37 5. White blood cell differential Monocyte Small lymphocyte Platelets Neutrophil Eosinophil Small lymphocyte Neutrophil Erythrocyte Young (band) neutrophil Monocyte Large lymphocyte Neutrophil Basophil

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39 5.1. Red blood cells biconcave discs and flexible plasma membrane but no nuclei or organelles

40 5.1. Red blood cells hemoglobin molecules oxygen carrying protein 4 chains of amino acids, each with a heme and a iron atom which is binding site for oxygen; CO2 carried also

41 Anemia definition: a decrease in number of red blood cells (RBCs) or less than the normal quantity of hemoglobin or a decrease in percentage of hematocrit in the blood symptoms: weakness, or fatigue, general malaise, pica, and sometimes poor concentration signs: pallor, jaundice consequence: hypoxia (lack of oxygen) in organs

42 Anemia anemia classification MCV: microcytic (iron deficiency), normocytic (acute blood loss, hemolysis), marcocytic anemia (VitB12 or folate deficiency) MCHC: hypochromic, normochromic, hyperchromic amemia (hereditary spherocytosis, sickle cell disease) causes: inadequate synthesis, increased destruction, loss

43 Anemia peripheral blood smear microcytic, hypochromic anemia (MCV< 80 fl, MCHC< 32 g/dl, Hb< 11 g/dl) Iron deficiency anemia

44 Anemia Anemia causes: inadequate erythropoiesis or hemoglobin synthesis Nutritional deficiency Iron-deficiency anemia VitB12- or folate-deficiency anaemia Bone marrow disorders Aplastic anemia Pure red cell aplasia

45 Anemia increased destruction of RBC (hemolytic anemia) membrane defects hereditary spherocytosis hereditary stomatocytosis paroxysmal nocturnal hemoglobinuria hemoglobin defects thalassemia sickle-cell anemia enzyme defect Glucose-6-phosphate dehydrogenase (G6PD) deficiency immune transfusion reaction hemolytic disease of the newborn nonimmune burn, sulfanilamide, lead poisoning, malaria infection

46 Anemia RBCs loss or dilution trauma chronic disease hookworm disease gastrointestinal ulcer haemorrhoids

47 Anemia hereditary spherocytosis (spherocytes)

48 Anemia hereditary stomatocytosis (stomatocytes)

49 Anemia thalassemia (tear drop cells) thalassemia (target cells)

50 Anemia sickle cell anemia (sickle cells) hemolytic anemia (schistocytes)

51 Anemia hemolytic anemia (schistocytes)

52 Anemia heinz bodies Heinz bodies appear as small blue round inclusions when stained with new methylene blue. They are composed of denatured hemoglobin, found in G6PD deficiency and thalassemia anemia.

53 Splenectomy howell-jolly bodies Howell-jolly bodies appear as round, purple inclusions in RBCs, and are composed of DNA, commonly seen in in patients with splenectomy.

54 Lead poisoning basophilic stipplings Basophilic stipplings appear as numerous, small purple inclusions in RBCs, and are aggregates of ribosomal RNA, most commonly seen in lead poisoning.

55 Ring form trophozoite of plasmodium vivax Anemia Ring form trophozoite of plasmodium falciparum Immature schizont of plasmodium vivax mature schizont of plasmodium vivax

56 5.2. Leukocytes (WBCs) granulocytes neutrophils with a multi-lobed (2-6) nucleus and very fine, neutral-stained cytoplasmic granules (enzymes) eosinophils large rosy-orange granules; bilobed nucleus basophils large, abundant, violet granules (obscure a large S-shaped nucleus) agranulocytes lymphocytes variable amounts of bluish cytoplasm (scanty to abundant); ovoid/round, uniform dark violet nucleus monocytes largest WBC; ovoid, kidney-, or horseshoe- shaped nucleus

57 Granulocytes 10 µm neutrophils eosinophil 10 µm basophil 10 µm

58 Agranulocytes lymphocyte 10 µm monocyte 10 µm

59 RBC neutrophil eosinophil Leukocytes basophil small lymphocyte monocyte

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62 Platelets Monocyte Neutrophils Lymphocyte

63 Toxic granulation neutrophil Increased basophilic granules in neutrophils, seen in severe infections, burns, malignancies, and pregnancy. Distinguish from basophils.

64 Dohle bodies neutrophil Sky blue inclusions in cytoplasm of neutrophils, seen in infections, burns, myeloproliferative disorders, and pregnancy. Composed of RER and glycogen granules.

65 platelets - small fragments of megakaryocyte cells 2-4 m diameter pseudopods contain granules 5.3. Platelets thrombocytosis: thrombosis thrombocytopenia: idiopathic thrombocytopenic purpura

66 5.3. Platelets Petechiae in patient with acute idiopathic thrombocytopenic purpura (AITP)

67 6. work calculate the number of white blood cells per milliliter in blood calculate the percent of 5 types of white blood cells in blood Cell Type Cell Number % Neutrophils Lymphocytes Monocytes Eosinophils Basophils Total 100 Draw a picture of formed elements you observed in blood smear slide

68 7. Case study A 56-year-old male farmer was evaluated for 2 months of black stools. He had fatigue and dizziness for 3 weeks with a weight loss. He denied abdominal pain, or diarrhea. No abnormal past history. He was afebrile. Physical examination is unremarkable except pallor. Laboratory studies show: RBC count: /µl ( /µl) Hemoglobin value: 7.0 g/dl (12-16 g/dl) Hematocrit: 24.8% (42-52%) MCV: 77.5 fl ( fl) MCHC: 28.2 g/dl (32-36 g/dl) Peripheral blood smear: many microcytic hypochromic RBCs Reticulocyte: 1.2%( %) WBC count: ( /µl) WBC differential: eosinophil was 6% (0.5-3%), other types of WBC were normal Platelet count: /µl ( /µl) Fecal occult blood (OB) test: strong positive Stool microscopy: numerous hookworm eggs Liver and renal function and tumor markers were normal What is the most likely diagnosis? Why?